Flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase (EC1.1.99.10; hereinafter, glucose dehydrogenase is also referred to as “GDH,” and FAD-dependent glucose dehydrogenase as “FAD-GDH”) is an enzyme that is mainly used for blood glucose concentration measurement and catalyzes the following reaction.D-glucose+Electron acceptor (oxidation type)→D-glucono-δ-lactone +Electron acceptor (reduction type)
Glucose oxidase is also known as an enzyme for quantifying blood glucose. However, this enzyme is said to have a problem in that glucose concentration measurement using glucose oxidase is affected by the concentration of dissolved oxygen because this enzyme may use molecular oxygen as an electron acceptor. Since glucose dehydrogenase is not influenced by such dissolved oxygen, it has been used as the main enzyme for glucose sensors in recent years. GDH includes FAD-dependent GDH, pyrroloquinoline quinone (PQQ)-dependent GDH, and NAD(P)-dependent GDH. PQQ-dependent GDH, such as Acinetobacter baumannii-derived PQQ-dependent GDH, has a problem with substrate specificity in that PQQ-dependent GDH is as reactive with maltose as it is with glucose. Examples of known NAD(P)-dependent GDH include Bacillus subtilis-derived NAD(P)-dependent GDH, Bacillus megaterium-derived NAD(P)-dependent GDH, Thermoproteus sp.-derived NAD(P)-dependent GDH, and the like. NAD(P)-dependent GDH has stricter substrate specificity than PQQ-dependent GDH. However, NAD(P)-dependent GDH is not necessarily useful since NAD(P), a coenzyme, needs to be added separately, thus requiring high costs in the production of reagents for quantifying glucose or sensors, as well as complexity in quality control. In that regard, FAD-GDH, such as FAD-GDH derived from the genus Aspergillus, is a coenzyme-bound type and has high substrate specificity; therefore, FAD-GDH has been found useful in recent years.
The production of glucose sensors involves the step of evaporating a GDH solution to dryness on a reaction layer. Attempts are often made to improve the production efficiency by performing heat treatment at 50° C. or more in this step to enhance the efficiency of evaporation to dryness. Although the heat treatment is effective for the production efficiency, proteins are generally known to be denatured by heat, and this risk increases especially in enzymes that have low thermal stability.
Moreover, usually, sensor strips after production are guaranteed for a maximum of two years at about room temperature. However, it is rare for general users using a glucose sensor to store sensor strips with strict temperature control. In particular, considering the situations in which summer temperatures are 35° C. or more, or sometimes exceed 40° C., it is easy to anticipate that high stability of the enzyme itself is desired. Enzymes having excellent thermal stability have commonly stable three-dimensional structures and can be said to be more suitable for long-term storage in harsh conditions.
Examples of known FAD-GDH include those derived from the genus Penicillium (Patent Literature 1), the genus Aspergillus (e.g., Patent Literature 2 and 3), the genus Mucor (e.g., Patent Literature 4 and 5), and the like. The upper limits of the heat resistance of all these enzymes in an aqueous solution are about 50° C. to 55° C., which are insufficient. Patent Literature documents 6 and 7 disclose an example in which the thermal stability of FAD-GDH was improved by using protein engineering technology. However, improvement in stability attained by modification using protein engineering technology has a limit, and thus high stability of the original wild-type enzyme is important.
None of the enzymes disclosed in the patent literature above withstands heat treatment at 60° C. to 65° C., and thus further improvement in stability is needed.